Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α(2)δ-1-induced downregulation of Ca(v)1.2 channels

Availability of voltage-gated calcium channels (Ca(v)) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Ca(v)1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Ca(v)1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Ca(v) channels. We co-expressed USPs and Ca(v)1.2 channels together with the accessory subunits β(2) and α(2)δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Ca(v) currents and the amount of Ca(v)1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α(2)δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Ca(v)1.2 and α(2)δ-1 subunits. Remarkably, α(2)δ-1, but not Ca(v)1.2 nor β(2), co-precipitated with USP2-45. These results suggest that USP2-45 binding to α(2)δ-1 promotes the de-ubiquitylation of both Ca(v)1.2 and α(2)δ-1 subunits, in order to regulate the expression of Ca(v)1.2 channels at the plasma membrane.